Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 19)

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See Appendix B for buffer formulations. Do not adjust pH.
4.3 Native PAGE Buffers
Running buffer (1x) 25 mM Tris, 192 mM glycine
Dilute 100 ml 10x stock (catalog #161-0734) with 900 ml diH
2
O.
Sample buffer 62.5 mM Tris-HCl, pH 6.8, 40% (w/v) glycerol, 0.01% (w/v) bromophenol blue
(catalog # 161-0738)
4.4 Sample Preparation
In the absence of SDS, the net charge of a polypeptide is determined by its amino acid composition
and the pH of the gel during electrophoresis, which is a function of the sample buffer, gel buffer, and
running buffer. Only polypeptides with a net negative charge migrate into gels under native conditions.
Most polypeptides have an acidic or slightly basic pI (~3–8). These proteins can be separated using the
following standard protocol:
1. Determine the desired protein concentration and load volume of your sample based on the
detection method used (see Chapter 10 for approximate stain sensitivities).
2. Dilute the sample with an equal volume of native sample buffer (do not heat the samples).
For example, combine: 5 μl sample
5 μl native sample buffer (catalog #161-0738)
10 μl total volume
Strongly basic proteins (pl >8.5) have a net positive charge and will not enter a Mini-PROTEAN TGX gel
under native conditions using Tris/glycine buffer. To allow polypeptides with a net positive charge
to migrate into a native gel, change the polarity of the electrodes by reversing the color-coded jacks
when connecting to the power supply.
4.5 Running Conditions
Running conditions for native PAGE are similar to the standard running conditions used for SDS-PAGE
(Section 3.4). If elevated temperature is a concern, run native PAGE at lower voltage; at lower voltages,
runs require more time to complete.
Table 4.1. Standard running conditions for native PAGE with one (1) gel in the Mini-PROTEAN Tetra cell . Run
conditions and times are approximate and assume a constant voltage of 200 V. When running more than one gel, current will
differ but temperature and run time should be close to those listed.
Current (mA) at 200 V
Gel Initial Final Temperature Run Time
1 Gel (buffer to 2-gel mark)
7.5%
35–37 17–20 28–30°C 38–40 min
10%
12%
4–15% 50–55 25–28 30–33°C 30–34 min
4–20%
Any kD
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